Pericytes are an understudied population of perivascular stromal cell associated with the microvasculature in most tissue, including the lung. Preliminary data in this grant application show that 1) FoxD1-derived, PDGFR+ putative pericytes express multiple MyD88-dependent Toll-like receptors, 2) following exposure to broncho-alveolar lavage (BAL) fluid collected from normal mice and mice with lung injury, PDGFR+ cells express inflammatory chemokines and downregulate Angpt1 via MyD88-dependent-signaling, and 3) PDGFR+ cells have similar transcriptional responses in vivo during lung injury. These data support the central hypothesis of this application that FoxD1-derived, PDGFR+ cells function as interstitial sentinels, which detect and res-pond to constitutive alveolar luminal molecules that are released into the interstitium following alveolar disruption. Activated PDGFR+ cells respond by promoting microvascular permeability and leukocyte recruitment. To test this hypothesis, three aims are proposed. The first aim will evaluate mechanism(s) by which PDGFR+ cells are activated by alveolar compartment molecules. Receptor(s) required for PDGFR+ cell activation by BAL fluid will be identified. PDGFR+ cell activating danger signals in BAL fluid will be identified, using a novel, unbiased approach termed orthogonal chromatography. The role of different PDGFR+ subpopulations will be evaluated. The second aim will use a unique 3D-microfluidic device with co-culture of primary endothelial cells to examine the role of PDGFR+ cells in regulation of endothelial function during inflammation, focusing on Angpt1-Tie2 signaling. Endothelial barrier integrity, leukocyte adhesion, and platelet adhesion will be measured. The third aim will determine in vivo functional consequences of pericyte activation during lung injury. The translated transcriptome of PDGFR+ cells during lung injury will be used to identify critical signaling networks and new functional responses of PDGFR+ cells. Lung injury will be compared between normal mice and mice with PDGFR-restricted MyD88 deletion. This project will test the hypothesis that PDGFR+ cells are unrecognized interstitial sentinel cells, which stand ready to detect and respond to significant alveolar epithelial injury and loss of barrier integrity. The acute respiratry distress syndrome (ARDS) or severe acute lung injury is a major cause of death and morbidity among critically ill patients, affecting an estimated 190,000 patients annually in the United States alone. Despite decades of research, the biology of ARDS is incompletely understood, and no effective pharmacological treatments exist. This project represents a new direction in the field with the potential to drive the development of novel, innovative therapeutic strategies.